MGcount: a total RNA-seq quantification tool to address multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts

Background Total-RNA sequencing (total-RNA-seq) allows the simultaneous study of both the coding and the non-coding transcriptome. Yet, computational pipelines have traditionally focused on particular biotypes, making assumptions that are not fullfilled by total-RNA-seq datasets. Transcripts from distinct RNA biotypes vary in length, biogenesis, and function, can overlap in a genomic region, and may be present in the genome with a high copy number. Consequently, reads from total-RNA-seq libraries may cause ambiguous genomic alignments, demanding for flexible quantification approaches. Results Here we present Multi-Graph count (MGcount), a total-RNA-seq quantification tool combining two strategies for handling ambiguous alignments. First, MGcount assigns reads hierarchically to small-RNA and long-RNA features to account for length disparity when transcripts overlap in the same genomic position. Next, MGcount aggregates RNA products with similar sequences where reads systematically multi-map using a graph-based approach. MGcount outputs a transcriptomic count matrix compatible with RNA-sequencing downstream analysis pipelines, with both bulk and single-cell resolution, and the graphs that model repeated transcript structures for different biotypes. The software can be used as a python module or as a single-file executable program. Conclusions MGcount is a flexible total-RNA-seq quantification tool that successfully integrates reads that align to multiple genomic locations or that overlap with multiple gene features. Its approach is suitable for the simultaneous estimation of protein-coding, long non-coding and small non-coding transcript concentration, in both precursor and processed forms. Both source code and compiled software are available at https://github.com/hitaandrea/MGcount.

Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq

Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.

First report of tomato fruit blotch virus infecting tomatoes in Brazil

Recently, a new blunervirus was reported in tomatoes showing fruit chlorotic lesions. This virus, named tomato fruit blotch virus (ToFBV), was found associated with the tomato fruit blotch disease in Italy and Australia, even though Koch’s postulates were not fulfilled and no viral particles were seen in leaf dips observed with an electron microscope (Ciuffo et al. 2020). In December 2019, symptoms of circular or irregular chlorotic blotches were observed in tomato fruits in an organic farm in Distrito Federal, Brazil. Five different tomato cultivars (2100 plants of cv. Sweet grape, 1700 of Giacomo, 560 of Grazianni, 160 of Tropical, and 160 of DRC 5640) were being grown in two greenhouses and all of them presented the symptoms in at least one fruit, particularly in older fruits. No virus-like symptoms were observed in young and middle leaves, but older leaves could not be examined because they were removed as a routine activity of the farm; and also due to the moderate infestation of the tomato russet mite Aculops lycopersici, associated with leaf and stem necrosis. No viral particles were observed in an electron microscope analysis of symptomatic fruit tissues, and sap inoculation and grafting of stems did not produce any symptom in indicator plants. Two young and asymptomatic plants with the first fruits still in development were removed from another greenhouse of the farm and transported to our greenhouse, but the typical blotch symptoms neither appeared in the fruits nor the necrosis symptoms in the leaves. Serological tests performed for all collected leaf and fruit samples using antibodies produced in-house against common tomato-infecting tospoviruses and potyviruses were negative, as well as a polymerase chain reaction (PCR) detection test for begomoviruses (Rojas et al. 1993). Total RNA from newly collected samples consisting of one symptomatic fruit sample and five asymptomatic leaf samples from distinct plants were individually extracted using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and pooled for next generation sequencing (NGS). The library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant (Illumina, San Diego, USA) and sequenced at Macrogen, Inc. (Seoul, South Korea) in an Illumina Novaseq6000 platform. The 4,621,977,958 reads obtained were trimmed using Trimommatic 0.35 (Bolger et al. 2014) and contigs were assembled using Velvet (Zerbino and Birney 2008). Following tblastx analysis on Geneious 9.1.8 (Biomatters Ltd.) and BLAST on the NCBI platform (Altschul et al. 1990), seven contigs matching tomato chlorosis virus (ToCV) and five contigs matching ToFBV were identified. Sequences for each of the four genome components of ToFBV (MK517477-MK517480) already present in databases were used as reference using the Map to Reference function in Geneious. A total of 338,402, 78,039, 555,302 and 461,474 reads mapped to virus genome components 1 to 4, respectively, with >99% coverage for each. Four final consensus sequences were used for BLAST analyses on NCBI and presented 97 to 99.7 % nucleotide identity with those used for mapping. These sequences were deposited in GenBank as isolate MAL under accession numbers MW546267 (RNA 1, 5770 nt), MW546268 (RNA 2, 3612 nt), MW546269 (RNA 3, 2826 nt) and MW546270 (RNA 4, 1950 nt). The primer pair Bluner1F (5’-ATTCCTGTTCCTTCGGATAAACTCGT-3’) and Bluner1R (5’-CACACGTGCAGGAAATGGAAAGA-3’) directed to RNA 1 was used to specifically detect the virus. Three leaf samples and two fruit samples, each from a different plant with typical symptoms, were tested positive for ToFBV and negative using ToCV-specific primers in RT-PCR (Dovas et al. 2002). This confirmed that although some plants pooled in the HTS library were infected with ToCV, the chlorotic blotch symptom was clearly associated with the presence of ToFBV. Furthermore, the ~0.5 kbp amplicon for ToFBV-specific primers from one randomly selected sample was sequenced with both primers and the resulting sequence shared 100% nt identity with the RNA 1 of ToFBV isolate Fondi2018 from Italy (MK517477). Then, the virus was detected in the tissue from the surface of another fruit, but not from its internal part, suggesting a superficial infection. The findings presented here are of high phytosanitary significance, given the strong symptoms associated with tomato fruit blotch disease and the identification of ToFBV in the tomato samples from Brazil.

cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing v2

Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios. However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability. We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. This protocol follows up after total RNA extraction (from the protocol at dx.doi.org/10.17504/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0). The subsequent cDNA library is prepared following the .

First report of grapevine virus L infecting grapevine in southeast France

Grapevine virus L (GVL) is a recently described vitivirus (family Betaflexiviridae) with a positive-sense single-stranded RNA genome. It has so far been reported from China, Croatia, New-Zealand, the United States and Tunisia (Debat et al. 2019; Diaz-Lara et al. 2019; Alabi et al. 2020; Ben Amar et al. 2020). It has significant genetic variability (up to 26% of nucleotide divergence between isolates) and the existence of four phylogroups has been proposed (Alabi et al. 2020). In the frame of a project investigating the possible links between grapevine trunk diseases and grapevine virome, viral high throughput sequencing (HTS)-based testing was performed on symptomatic and asymptomatic grapevines collected in July 2019 in vineyards of four areas in France (Bourgogne, Charentes, Gard, Gironde) corresponding to five cultivars of Vitis vinifera (Cabernet franc, Cabernet Sauvignon, Chardonnay, Sauvignon, Ugni blanc). Total RNAs were purified from powder of 105 trunk wood samples using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and RNA-seq libraries were prepared using Zymo-Seq RiboFree Total RNA Library Prep Kit (Ozyme, Saint Cyr l’Ecole, France). HTS was performed on a S4 lane of Illumina NovaSeq 6000 using a paired-end read length of 2x150 bp. The trimmed sequence reads obtained from Chardonnay plants CH30-75M (99.9 M) and CH37-19S (114 M) from a vineyard in Gard were analyzed using CLC Genomics Workbench v21 (Qiagen, Courtaboeuf, France) and revealed complex mixed infections. Besides contigs representing a complete GVL genome (average scaffold coverage: 6,197x and 2,970x, respectively), contigs from grapevine rupestris stem pitting virus (1,697x ; 1,124x), grapevine virus A (82x ; 95x), grapevine pinot gris virus (1,475x ; 866x), grapevine leafroll-associated virus 3 (5,122x ; 1,042x), hop stunt viroid (13,783x ; 29,514x) and grapevine yellow speckle viroid 1 (690x ; 1158x) were also identified. Plant CH37-19S was also co-infected by grapevine rupestris vein feathering virus (164x). The GVL contigs integrated respectively 320,000 and 152,000 reads (corresponding to 0.32% and 0.11% of filtered/trimmed reads, respectively). The GVL genomic sequences from each sample (7,616 nt) have been deposited in GenBank (Accession nos. OK042110 and OK042111, respectively). The two contigs are nearly identical (99.9% nt identity) and share respectively 97.5% and 95.9% with GVL-KA from the USA (MH643739) and GVL-RS from China (MH248020), the closest isolates present in GenBank. To confirm the presence of GVL, the original grapevines were resampled in the field and total RNAs were extracted as described above from cambial scrappings and leaves. Total RNAs were used for RT-PCR tests using primers targeting a 279-bp fragment corresponding to the 3’ end of the coat protein gene and part of the nucleic acid binding protein gene (Debat et al. 2019). The Sanger-derived sequences from the amplicons shared 100% nt identities with the corresponding sequences of the HTS assembled genomes, confirming the presence of GVL in both tissues of both grapevine samples. To our knowledge, this represents the first report of the occurrence of GVL in vineyards in France. Given the complex mixed infection present in the two analyzed grapevines, no conclusions can be drawn on the pathogenicity of GVL. Further efforts are needed to better understand GVL distribution and its potential pathogenicity to grapevine. References Alabi, O J., et al. 2020. Arch. of Virol. 165:1905-1909. Ben Amar, A., et al. 2020. Plant disease 104:3274. Debat, H., et al. 2019. Eur J Plant Pathol. 155:319. Diaz-Lara, A., et al. 2019. Arch. of Virol. 164:2573. Acknowledgments The authors are grateful to the “Plan National Dépérissement du Vignoble” (Mycovir project) for the financial support

A lyophilized form of xenogeneic total RNA stimulates hematopoiesis in post-radiation myelosuppression

Введение. Аллогенная суммарная РНК, выделенная из клеток лимфоидных органов, стимулирует регенерацию кроветворной ткани после острого и хронического нарушения кроветворной функции. Цель исследования: 1) доказательство отсутствия ксеногенных ограничений механизмов лимфоцитарного контроля регенеративных процессов на примере гемостимулирующего действия суммарной РНК лимфоцитов бычьей селезенки в отношении кроветворения крыс, подвергшихся гамма-облучению в сублетальной дозе; 2) сравнительный анализ эффективности нативной и лиофилизированной форм указанной РНК. Методика. Работа выполнена на белых нелинейных крысах-самцах массой 200-220 г. Cуммарную РНК выделяли методом фенол - хлороформной экстракции из лимфоидных клеток бычьей селезенки. Для создания исходной миелосупрессии 30 крыс подвергли однократному общему воздействию гамма-излучения с источником 60Co в дозе 6 Гр при мощности дозы 0,1 Гр/с, после чего разделили их на 3 равные группы. Через 2 ч после облучения крысам контрольной группы внутрибрюшинно ввели по 0,5 мл 0,9% NaCl; крысам 2-й группы - нативную суммарную РНК в дозе 30 мкг/100г массы, крысам 3-й группы - лиофилизированную суммарную РНК в аналогичной дозе. На 3-и, 7-е и 12-е сут в периферической крови определяли количество ретикулоцитов, лейкоцитов и тромбоцитов, после чего крысы были выведены из эксперимента с целью исследования костномозгового кроветворения. Через 12 сут в костном мозге определяли количество эритроидных, лимфоидных, мегакариоцитарных и миелоидных клеток. Из костного мозга выделяли эритробластические островки (ЭО) и дифференцировали их на пролиферирующие (ЭО 1,2 классов и реконструирующиеся ЭО) и зрелые (ЭО 3 класса и инволюциирующие ЭО) морфо-функциональные клеточные ассоциации. Результаты. Под влиянием ксеногенной суммарной РНК в периферической крови крыс в 2-3 раза увеличилось количество лейкоцитов и в 1,6-1,75 раза возросло число ретикулоцитов. В костном мозге увеличилось количество пролиферирующих миелоидных и лимфоидных элементов, а также общее число клеток эритроидного ряда. Ксеногенная суммарная РНК стимулировала образование ЭО как на основе контакта свободных костномозговых макрофагов с молодыми эритроидными клетками (ЭО 1 и 2 классов), так и на базе реконструкции (ЭО реконструирующиеся). Сравнительный анализ эффектов нативной и лиофилизированной суммарной РНК не выявил различий между гемопоэтическими показателями у крыс, получивших эти препаратов. Заключение. Суммарная РНК, выделенная из лимфоидных клеток бычьей селезенки, активирует гемопоэз у крыс с постлучевой миелосупрессией, что свидетельствует об отсутствии ксеногенных ограничений у млекопитающих в механизмах лимфоцитарного контроля восстановительных процессов. Лиофилизированная суммарная РНК активирует костномозговое кроветворение в те же сроки и в том же объеме, что и нативная форма. Introduction. Allogeneic total RNA isolated from cells of lymphoid organs stimulates regeneration of hematopoietic tissue after acute and chronic disturbance of hematopoietic function. Aim. 1) To prove the absence of xenogeneic limitation for the lymphocytic regulation of regenerative processes using an example of the hemo-stimulating effect of total RNA from bovine spleen lymphocytes on hematopoiesis in rats exposed to sublethal gamma-irradiation; 2) To perform a comparative analysis of the effectiveness of the native and lyophilized forms of the total RNA. Methods. Experiments were performed on white outbred male rats weighing 200-220 g. Total RNA was isolated from bovine spleen lymphoid cells by phenol-chloroform extraction. To create the initial myelosuppression, 30 rats were exposed to a single general 60Co gamma radiation (6 Gy at 0.1 Gy/s). The rats were then divided into 3 equal groups. Two hrs after irradiation, the rats of the control group were injected intraperitoneally with 0.5 ml of 0.9% NaCl; rats of the second group received native total RNA, 30 μg/100 g body weight, and rats of the third group received lyophilized total RNA at a similar dose. On days 3, 7, and 12, the number of peripheral blood reticulocytes, leukocytes, and platelets was measured. The rats were then sacrificed, and bone marrow hematopoiesis was studied. After 12 days, the number of bone marrow erythroid, lymphoid, megakaryocytic, and myeloid cells was measured. Erythroblastic islets (EIs) were isolated from the bone marrow and differentiated into proliferating (class 1 and 2 EIs and reconstructing EIs) and mature (class 3 EIs and involving EIs) morpho-functional cell associations. Results. Under the influence of xenogeneic total RNA, the number of peripheral blood leukocytes increased by 2-3 times, and the number of reticulocytes increased by 1.6-1.75 times. In the bone marrow, the number of proliferating myeloid and lymphoid cells increased, as did the total number of erythroid cells. Xenogeneic total RNA stimulated formation of EIs, based both on the contact of free bone marrow macrophages with young erythroid cells (class 1 and 2 EIs) and on reconstruction (reconstructing EIs). Comparative analysis of the effects of native and lyophilized total RNA did not reveal differences between hematopoietic parameters in rats that received these agents. Conclusion. Total RNA isolated from bovine spleen lymphoid cells activates hematopoiesis in rats with post-radiation myelosuppression. This indicates the absence of mammalian xenogenic limitation of lymphocytic control of recovery processes. Lyophilized total RNA activates bone marrow hematopoiesis at the same rate and to the same extent as the native form.

Single-cell quantification of a broad RNA spectrum reveals unique noncoding patterns associated with cell types and states

The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current single-cell RNA-sequencing (RNA-seq) methods are unable to simultaneously monitor all forms of RNA transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and noncoding RNA from a single cell. Smart-seq-total does not require splitting the RNA content of a cell and allows the incorporation of unique molecular identifiers into short and long RNA molecules for absolute quantification. It outperforms current poly(A)-independent total RNA-seq protocols by capturing transcripts of a broad size range, thus enabling simultaneous analysis of protein-coding, long-noncoding, microRNA, and other noncoding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T, and MCF7 cells, as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. By analyzing the coexpression patterns of both noncoding RNA and mRNA from the same cell, we were able to discover new roles of noncoding RNA throughout essential processes, such as cell cycle and lineage commitment during embryonic development. Moreover, we show that independent classes of short-noncoding RNA can be used to determine cell-type identity.

First Report of Schlumbergera virus X in Dragon Fruit (Hylocereus spp.) in Spain

Dragon fruit (Hylocereus undatus) is a high-value fruit crop, introduced about a decade ago in the mainland of Spain. In 2021, chlorotic spots were observed on young cladodes in a commercial dragon fruit orchard in the province of Seville (southern Spain). Sap extracts from 4 symptomatic cladodes were used to mechanically inoculate indicator plants: no symptoms were produced in Datura stramonium plants, but Chenopodium amaranticolor reacted with chlorotic local lesions and prickly pear plants (Opuntia ficus-indica) showed irregular yellow ringspot symptoms on young cladodes at 30 days post inoculation. Total RNA was extracted from all 4 symptomatic cladodes as previously described (Pallas et al. 1987). Reverse transcription (RT)-PCR, which was carried out with M-MLV-RT and Go Taq Pol (Promega Biotech Ibérica, SL, Madrid, Spain) and tobamovirus primers (Dovas et al. 2004), failed to produce any amplicons. Electrophoretic analysis of dsRNA, extracted from symptomatic cladodes, yielded a banding pattern similar to the one reported for potexviruses (Valverde et al. 1986). Primers specific for Cactus virus X (Kim et al., 2016) failed to produce amplicons, whereas potexvirus group primers (Potex F5/Potex R2) (van der Vlugt and Berendsen 2002), amplified an expected 584-bp amplicon from RNA extracts of all 4 field-collected samples. The RT-PCR products from the four samples were Sanger-sequenced. All showed identical sequence results (GenBank Accession MZ614940) with a predicted amino acid identity of 99% with the corresponding RNA-dependent RNA polymerase amino acid sequence of Schlumbergera virus X (SchVX) (GenBank Accession No. ACD99908). SchVX-specific primers (431s, 5‘-TTTGAGGAGTTCGTCAGCAAGA-3‘ and 431As, 5‘-TCAAGAGCCCATTGAGAGAGTG-3‘) that were designed based on the new sequence, amplified the expected amplicon of around 430 nucleotides from the total RNA extracts of the four samples. The amplicons were Sanger-sequenced and the expected nucleotide sequence was obtained. This pair of primers were used in RT-PCR tests on subsequent surveys in 2 commercial dragon fruit greenhouses from the province of Seville, and in 1 experimental greenhouse in the province of Almeria. All samples from 25 symptomatic plants of H. undatus, H. hybridum, H. costaricensis, and H. purpusii in Seville and from 1 symptomatic H. undatus plant from Almeria tested positive for SchVX, while 15 asymptomatic plants tested negative. The results obtained in this investigation support that SchVX is present in the cladodes of dragon fruit plants expressing the symptoms. SchVX has been reported previously from H. undatus from Brasil (Duarte et al. 2008) and from prickly pear in Mexico (De La Torre-Almaráz et al. 2016), and to our knowledge, this is the first report of the virus in Spain. These findings suggest that SchVX has been introduced in dragon fruit farms from Spain and propagation of this emerging crop through planting of cuttings should include testing for this virus in order to prevent further spread.

Compartment-specific total RNA profile of Hippocampal and Cortical cells from Mesial Temporal Lobe Epilepsy tissue

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disease characterized by recurrent seizures. The pathogenic mechanisms underlying mTLE involve defects in post-transcriptional regulation of gene expression. So far, transcriptome profiles from epileptic tissue have been generated using whole cells, thereby lacking information on RNA localization and function at a subcellular level. In line with this, we have previously observed by in situ hybridization that a few microRNAs (miRNAs) display subcellular mis-localization with aberrant enrichment in the nucleus in human hippocampal mTLE tissue samples (Kan et al., 2012). To further investigate the possible mechanisms leading to the mis-localization of miRNAs, we set out to understand the compartment-specific total RNA (coding and non-coding) profile of human mTLE tissue samples. For this, we have successfully established a protocol to isolate cytoplasmic and nuclear compartments from human hippocampal tissue. After confirming the purity of the isolated cell compartments, we performed total RNA-sequencing (RNA-seq) on five resected hippocampal (HC) mTLE (no hippocampal sclerosis (non-HS)) samples and five HC postmortem control samples. Similarly, six neo-cortical (Cx) tissue samples from mTLE non-HS and HS International League Against Epilepsy (ILAE) Type 1, or mTLE+HS, samples were compared with six Cx postmortem controls. Our dataset provides a comprehensive overview of compartment-specific transcriptomic profiles of pharmacoresistant mTLE patient HC and Cx tissue, which in further studies can be used to investigate disease mechanisms.